While it is well known that low affinity (approximately >1 uM) antibodies frequently bind multiple antigens, the natural and man-made processes of affinity maturation and optimization (directed evolution or molecular evolution) are typically designed to increase both the affinity and specificity to only a single epitope of a molecule at high affinity. In general, for most applications, specificity is an important attribute; for example, in therapeutics specificity can prevent off-target effects that are likely to decrease the safety of a molecule. Nevertheless, there is substantial utility in the ability to bind a limited number (for example 2, 3, 4, 5, 6, 7, 8, 9, or 10, but preferably 2 or 3) of selected target antigens, particularly for example for treating diseases where there are multiple activation pathways, such as diseases related to cancer. Immunotherapy for cancer with regular monoclonal antibodies does not activate T-cells, because they do not express Fc-receptors. Bi-specific antibodies (one arm binds the tumor marker, one arm binds the T-cell specific surface antigen, e.g. CD3) can overcome this problem and link tumor cells and T-cells. In addition, tri-functional antibodies (IgGs with 2 different binding specificities and an intact Fc domain) can also bind to Fc receptor expressing cells like macrophages and dendritic cells. The tumor cell is then connected to one or two cells of the immune system, which subsequently destroy it.
Several groups have sought to design bi-specific antibodies, for example by making heterologous antibodies through uncoupling of the antibody heavy chains of two independent antibodies through disulfide bond reduction and then reassociating the antibodies under an oxidizing environment so that the Fab's of the bivalent IgG molecule are different and bind to separate antigens. This approach has the disadvantage of having no avidity to the identical target molecule and yields a costly product generation and purification process. Other schemes have also been developed that include sequence expansion within the Fab, effectively duplicating the antigen binding pocket on the antibody so that each binding pocket can have different antigen specificity on a single antibody molecule. While this simplifies manufacturing and purification, the structure is foreign to the human body and runs a risk of stimulating a negative immune reaction in a patient. Still others have employed novel covalent linkages to achieve multi-epitope binding, creating “antibody-like” molecules.
Traditional bi-specific or multi-specific antibodies can be difficult to manufacture. For example, these antibodies can be constructed by expressing two separate heavy and two separate light chains in the same cell (Quadroma technology, Milstein et al., 1983), however, this approach is problematic because in addition to the desired light chain1/heavy chain1—heavy chain2/light chain2 hetero-dimer, all 10 possible heavy chain and light chain combinations will be formed (Suresh et al., 1986). The binding affinity and specificity of unwanted light chain/heavy chain pairings is unknown. Efforts to reduce the complexity of the possible light chain/heavy chain assemblies of the resulting populations includes methods such as the “knob in hole” design (Ridgeway et al., 1996, incorporated herein by reference) where the Fc part of the heavy chains can be modified to eliminate the formation of some of the homo-dimers. However, the populations are still very complex with traditional technologies, even with these modifications. The desired bispecific (or multispecific) product is only a small fraction of the mixture, making the purification of the bispecific (multispecific) antibody difficult and sometimes not feasible on a commercial scale in many cases.
Multi-specific antibodies of the present invention are distinguished by their ability to bind to multiple antigens with specificity and with affinity (for example, <10 nM). In one aspect, the multi-specific antibodies of the present invention comprise two different heavy chain variable domains (binding two or more different antigens), a single light chain variable domain that fits both heavy chain variable domains or has been optimized to fit both heavy chains, and an Fc that forms heterodimers or has been optimized to form heterodimers. Construction of multi-specific antibodies of the present invention can be accomplished in several ways. For example, in one approach, the variable domains of two parent monoclonal antibodies are evolved using one of several methods, including methods described herein, so that the same single light chain can functionally complement both heavy chains from the parent antibodies. One can also evolve the heavy chain of a single parent antibody so that it can bind to a second target, creating a new heavy chain, followed by pairing the new heavy chain with the light chain from the parent antibody. In yet another approach, a light chain of a single parent antibody can be evolved so that it can bind a second target, creating a new light chain, followed by pairing the new light chain with the heavy chain from the parent antibody. The Fc portion that forms heterodimers of the multi-specific antibodies of the present invention can be created using a “knob-in-hole” type approach, or any other approach that motivates the Fc to form or results in the Fc forming heterodimers.
Examples of construction of multi-specific antibodies of the present invention are further described herein.
In one embodiment, following isolation of multi-specific antibodies of the present invention, the affinity of a multi-specific antibody to one or more antigens or targets can be further improved through an evolution process, for example a comprehensive evolution process. In one aspect of the comprehensive evolution process, up-mutants are identified during screening as those mutants improving binding to at least one or both antigens without causing a decrease in binding to the alternative antigen. These up-mutants (changes can be in both the heavy and/or light chains) can then be further mixed and matched, combinatorially, for example.
In certain other instances, a lower affinity to one of the target antigens and a higher affinity to another target antigen is desirable. For example, Y. Joy Yu, et al published in Science Translational Medicine, 25 May 2011, Vol 3, Issue 84 84ra44, “Boosting Brain Uptake of a Therapeutic Antibody by Reducing Its Affinity for a Transcytosis Target” that a bispecific antibody with one arm comprising a low-affinity anti-transferrin receptor antibody and the other arm comprising a high-affinity BACE1 antibody was able to cross the blood brain barrier and reach therapeutic concentrations in the mouse brain. This bispecific antibody was substantially more effective compared to a parent monospecific antibody.
Thus, in another aspect of the comprehensive evolution process, up-mutants are identified during screening as those mutants improving binding to one antigen. Although the mutants that cause a decrease in binding affinity to a second antigen are not prioritized, they could be useful in situations where in combinatorial fashion they lose their inhibitory effect when combined with other mutations during the combination of mutations process. Screening can be performed to identify lead candidates based upon their overall affinity as well as their respective on and off rates for antigen:antibody binding to each of the chosen antigens.
Multi-specific antibodies of the present invention may also be optimized for increased or decreased activity or stability in other conditions, such as pH, oxidation, temperature, pressure, or different ion concentrations.